The present invention relates to purified and stabilized isoforms of Na,K-ATPase and methods of obtaining same.
For over two hundred years the plant-derived digitalis glycosides have been used to treat congestive heart failure. Digoxin, the principal cardiac glycoside (CG) used in clinical practice, has been shown to reduce mortality from congestive heart failure (CHF). However, digoxin has a narrow therapeutic window (particularly in K-depleted patients) and has been shown to induce cardiac arrhythmias.
The mechanism of the positive inotropic effects of cardiac glycosides involves (a) inhibition of the cardiac Na,K-ATPase, (b) reduction of the normal trans-membrane Na gradient (c) functional coupling with the 3Na/Ca exchanger to increase Ca entry into the cell (d) pumping of the excess Ca into the sarcoplasmic reticulum (SR) by the Ca-ATPase (e) release of Ca from SR via ryanodine receptor (RyR) and IP3 Ca channels upon electrical excitation of the muscle and (f) increased force of contraction of cardiac muscle due to the raised cytoplasmic Ca concentration.
The Na,K-ATPase consists of both α and β subunits. Four isoforms of a (α1, α2, α3 and α4) and three isoforms of β (β1, β2 and β3) are known. Human heart is known to express not only the ubiquitous α1 “house-keeping” isoform but also the α2 and α3 isoforms. In addition, the human heart is known to express β1 but not β2. This indicates that in humans, the pump complexes are α1,β1 α2,β1 and α3β1.
It is believed that CG toxicity results from excessive inhibition of Na,K-ATPase, resulting in Ca accumulation in the cell, beyond the capacity of the SR to sequester it. This is termed Ca-overload. As a result of the Ca-overload, the 3Na/Ca exchanger (NCX1) is activated, and because this exchanger is electrogenic the membrane potential is partially depolarized following the normal repolarization phase of the action potential. This is termed delayed after depolarizations (DADS). DADS induce spontaneous firing and unsynchronized heart beats—i.e arrhythmias. Since the α1 isoform is the major form in human heart, while α2 (and α3) are present at lower levels, excessive inhibition is inevitably associated with the housekeeping α1 isoform. Therefore, by avoiding inhibition of α1, and restricting inhibition to a minor fraction of the Na,K-ATPase molecules, excess inhibition of Na,K-ATPase and Ca-overload should be automatically avoided. As mentioned above, in human heart both α2 and α3 isoforms are expressed as well as α1. Thus, in principle, either an α2 or an α3-specific inhibitor could reduce the Ca-overload problem and be a safer inotropic drug.
CG toxicity is exacerbated in patients with depleted serum K, associated with congestive heart failure or following diuretic therapy. Excessive inhibition of the Na,K-ATPase can be the result of diminution of the normal CG-serum K antagonism, which determines the appropriate therapeutic dose of CG (1-2 nM for digoxin). Another aspect of CG toxicity is the fact that CG's dissociate slowly from the Na,K-ATPase, so that the effect of toxic concentrations is not readily reversed. New compounds in which these features differ from those of digoxin, the standard CG in clinical use, either for both isoforms, or in an isoform-specific way, could also be important in the search for a safer CG.
Recent research suggests that in addition to pumping ions, Na(+)/K+-ATPase interacts with neighboring membrane proteins and organized cytosolic cascades of signaling proteins to send messages to the intracellular organelles [Xie and Askari, 2002, Eur J Biochem, 269, 2434-2439]. The likely extracellular physiological stimuli for the signal transducing function of Na+/K+-ATPase are the endogenous ouabain-like hormones, and changes in extracellular K+ concentration. The signaling pathways that are rapidly elicited by the interaction of ouabain with Na(+)/K(+)-ATPase are reported to be independent of changes in intracellular Na(+) and K(+) concentrations, and involve a cascade of events via the EGF receptor/Src and the ERK1/p42/44 MAPK pathways to regulate growth-related gene transcription. It has also been suggested that this pathway is necessary for the inotropic effect of CG's by increasing cytoplasmic Ca (Tian et al., 2001, Am J Physiol Heart Circ Physiol, 281, H1899-1907). Thus, this ouabain-signaling pathway is of interest in relation to long-term growth regulation and cardiac hypertrophy, and in this respect could be another target of novel CG's.
A unique mode of regulation of the Na, K-ATPase has been described recently, involving interactions between the α/β complex and the FXYD proteins. The FXYD family consists of seven short single span transmembrane proteins (N terminus extracellular), named after the conserved FXYD motif in the extracellular segment. The FXYD proteins show a tissue-specific expression pattern. Six members of the family, FXYD1 (PLM), FXYD2 (γ), FXYD3 (Mat-8), FXYD4 (CHIF), FXYD5 (RIC), and FXYD7, are now known to interact specifically with the Na,K-ATPase, and modulate the functional properties. The FXYD proteins are not required for basic pump function, but they act as accessory subunits to the α and β subunits, and modulate the kinetic properties in a tissue-specific fashion.
Previous work with human isoforms of Na,K-ATPase expressed in Xenopus Oocytes showed that H3-ouabain binds to and dissociates from α2 much faster than to α1 and α3 while equilibrium dissociation constants are fairly similar. Human isoforms have also been expressed in the yeast S. cerevesiae (Muller-Ehmsen et al., American J. Physiol Cell Physiol, 281: c1355-C1364, 2001), where roughly similar dissociation constants for α1, α2 and α3 for ouabain were measured. A low expression level of α2 was observed which was explained by instability. Neither the X oocyte nor the S. cerevesiae expression systems provide suitable material for large scale screening of inhibitors since the density of the induced Na,K-ATPases was not sufficiently high for purification and thus the induced protein is unstable (α2) (S. Cerevesiae), or the electrophysiological measurements are too unwieldy (X. Oocytes). In order to carry out high throughput screens of inhibitors on the different human isoforms, and compare inhibitor properties in detail, availability of purified active and stable Na,K-ATPase isoforms is required.
Porcine Na,K-ATPase has been expressed in the methanotrophic yeast P. pastoris (Cohen et al., 2005, J Biol Chem, 280, 16610-16618; Strugatsky et al., 2003, J Biol Chem, 278, 46064-46073). The present inventors showed (Cohen et al., 2005, J Biol Chem, 280, 16610-16618) that following expression of Porcine α/his10-β subunits and solubilization of the yeast membranes in n-dodecyl-β-maltoside (DDM), detergent-soluble α/his10-β complexes could be purified utilizing a combination of Ni2+-NTA bead chromatography and size-exclusion HPLC. The procedure produced 70-80% percent pure porcine Na,K-ATPase. This complex, two-step procedure however is not convenient for producing large quantities of Na,K-ATPase for high through-put screening.
There remains a widely recognized need therefore, and it would be highly advantageous to have simpler methods of expressing and purifying different isoforms of Na,K-ATPases, where the resultant Na,K-ATPases are functional and stable over an extended period of time. In particular it would be advantageous to have purified human isoforms for testing selectivity of inhibitors, since native human tissue is not readily available and cardiac tissue is not suitable for this task because it comprises a mixture of the isoforms.